Summary

Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex
process that involves multiple anatomical sites. Once HSC precursors have been
specified from mesoderm, they have to mature into functional HSCs and undergo
self-renewing divisions to generate a pool of HSCs. During this process,
developing HSCs migrate through various embryonic niches, which provide
signals for their establishment and the conservation of their self-renewal
ability. These processes have to be recapitulated to generate HSCs from
embryonic stem cells. Elucidating the interactions between developing HSCs and
their niches should facilitate the generation and expansion of HSCs in vitro
to exploit their clinical potential.

Introduction

The lifelong production of blood cells depends on hematopoietic stem cells
(HSC) and their ability to self-renew and to differentiate into all blood
lineages (Weissman, 2000).
The original pool of HSCs is formed during embryogenesis in a complex
developmental process that involves several anatomical sites (the yolk sac,
the aorta-gonadmesonephros region, the placenta and the fetal liver), after
which HSCs colonize the bone marrow at birth (Figs
1,
2). During postnatal life, a
steady state is established in which HSC pool size is maintained by the
regulation of HSC self-renewal and differentiation. This is possible because
the bone marrow contains specialized niches in which the multipotency of HSCs
is conserved through cell divisions, while their progeny are directed towards
lineage differentiation (Wilson and
Trumpp, 2006). During homeostasis, most adult HSCs are quiescent
and divide only rarely to maintain an appropriate quantity of differentiated
blood cells and to renew the HSC pool
(Cheshier et al., 1999). HSC
pool maintenance and concomitant lineage differentiation are facilitated
either by asymmetric selfrenewal in which specific cell fate determinants are
redistributed unequally to the two daughter cells; or via environmental
asymmetry, in which one daughter cell leaves the niche that sustains HSC
selfrenewal and is then exposed to an environment that promotes lineage
differentiation (Wilson and Trumpp,
2006). Interestingly, all LTR-HSCs (long-term reconstituting HSCs)
in the adult bone marrow that can engraft a recipient upon transplantation are
in G0 phase (Passegue et al.,
2005). Thus, although HSCs have to divide in order to self-renew,
their cell division can only be safely completed within a correct niche;
otherwise, their engraftment ability and survival is greatly challenged.

It has been a long-standing challenge to recapitulate ex vivo the correct
micro-environment that supports HSC self-renewal. Cultured HSCs rapidly lose
their ability to engraft and self-renew in vivo, which limits the options to
maintain, expand or manipulate HSCs in vitro for therapeutic purposes.
Likewise, attempts to use mouse or human embryonic stem (ES) cells to generate
HSCs that can permanently reconstitute the hematopoietic system of the
recipient have not been successful, although differentiated hematopoietic
cells can be generated with relative ease
(Keller, 2005;
Kyba and Daley, 2003;
Wang et al., 2005a). So far,
robust and sustained multi-lineage reconstitution of adult hematopoiesis from
mouse ES cell-derived hematopoietic precursors has been obtained only by
overexpressing the transcriptional regulators Hoxb4 and Cdx4 in mouse ES cells
(Kyba et al., 2002;
Wang et al., 2005b). Although
these studies highlight important principles about the transcriptional
programs that regulate key properties of definitive HSCs, a safer approach to
generating HSCs for therapeutic applications would be to provide the
appropriate signals from the environment where HSCs develop. In order to
generate HSCs from ES cells, hematopoietic precursors have to go through the
same developmental process as they do during embryogenesis. Thus, to learn how
`stemness' of HSCs can be established and maintained, it is crucial to define
the cellular niches and key signals that support HSCs at each stage of
development.

The challenge of fetal hematopoiesis is to generate differentiated blood
cells that are immediately required for embryonic growth and development, and
to establish concomitantly a stockpile of undifferentiated HSCs, even though
the bone marrow and its specialized niches have not yet developed.
Consequently, multiple anatomical sites participate in fetal hematopoiesis
(Fig. 1). The shift of
hematopoiesis from one location to another is required as the anatomy of the
embryo changes during organogenesis. Furthermore, compartmentalizing fetal
hematopoiesis into multiple sites might allow different inductive signals from
the micro-environments to support the development of undifferentiated HSCs,
while concomitantly generating mature blood cells in another location. The use
of multiple fetal hematopoietic sites is common to many species, such as
flies, amphibians, fish, birds, rodents and humans
(Ciau-Uitz et al., 2000;
Tavian and Peault, 2005b;
Traver and Zon, 2002). To
date, HSC development has been characterized in most detail in the mouse,
which, in many respects, serves as a model for human hematopoiesis
(Tavian and Peault, 2005a;
Tavian and Peault, 2005b;
Tavian and Peault,
2005c).

Embryonic hematopoiesis in mice starts after gastrulation, when a subset of
specialized mesodermal precursor cells commit to becoming blood cells
(Fig. 2). The first precursors
migrate to the yolk sac to initiate embryonic red blood cell production,
whereas definitive HSCs, which possibly originate from a different subset of
mesodermal cells, develop in a different location (reviewed by
Jaffredo et al., 2005).
Importantly, the developing HSCs are required to complete a maturation process
that permits their engraftment and survival in future hematopoietic niches.
Furthermore, the initial HSC pool that emerges from hemogenic sites must
expand to establish an adequate supply of HSCs for postnatal life.
Consequently, fetal HSCs are largely cycling, and have to undergo symmetric
cell divisions, in which both daughter cells retain self-renewal ability and
multipotency, resulting in a net expansion of HSCs
(Lessard et al., 2004). As
fetal HSCs are markedly different from adult HSCs with respect to their cell
cycle status and proliferative capacity, it is conceivable that different
mechanisms control engraftment and selfrenewal of HSCs during fetal and adult
life.

Multiple studies have documented that the AGM (aorta-gonadmesonephros)
region (see Figs 1,
2), which consists of the
dorsal aorta, its surrounding mesenchyme and the urogenital ridges, is a
source of definitive HSCs (Cumano et al.,
1996; Godin and Cumano,
2002; Medvinsky and Dzierzak,
1996; Muller et al.,
1994). Additionally, HSCs are formed in umbilical and vitelline
arteries (de Bruijn et al.,
2000). These HSCs probably colonize the developing fetal liver,
which serves as the main organ for HSC expansion and differentiation until
late fetal life, at which time bone marrow hematopoiesis is established.
However, it has been questioned whether AGM-derived HSCs alone can supply the
rapidly growing fetal liver HSC pool
(Kumaravelu et al., 2002), or
whether the yolk sac also supports the formation, maturation or expansion of
HSCs later during development. Additional reservoirs for HSCs have been sought
by mapping the anatomical distribution of HSCs in the embryo. Yet, no
significant pools of HSCs have been found in the embryo proper
(Kumaravelu et al., 2002). A
novel perspective to HSC development came with the discovery that the murine
placenta harbors a large pool of multipotential progenitors and HSCs during
midgestation, indicating that the placenta has an important role in the
establishment of HSCs (Alvarez-Silva et
al., 2003; Gekas et al.,
2005; Ottersbach and Dzierzak,
2005).

Hematopoietic organs in mouse and human embryos. An illustration of
(A) a mouse and (B) a human embryo, showing the hematopoietic
organs at 11 days and 5 weeks of gestation, respectively (yellow, yolk sac;
green, dorsal aorta; red, fetal liver; blue, umbilical vessels and fetal
vasculature in the placenta). The hematopoietic function of the human placenta
has not yet been experimentally proven. AGM, aorta-gonad-mesonephros
region.

Despite the progress in identifying and quantifying HSCs in fetal organs,
little is known about the origins of the different HSC pools and their
relative contributions to adult hematopoiesis. It has been difficult to trace
HSC precursors in mammalian embryos at earlier developmental stages when these
cells express different surface markers compared with mature HSCs, and when
they may not yet score in conventional transplantation assays. To facilitate
the characterization of these developmental processes in the embryo, lineage-
and developmental stage-specific mouse models have been generated
(Gothert et al., 2005), and
functional assays for immature HSC precursors have been improved
(Table 1). Furthermore, our
knowledge of the micro-environmental signals that support HSCs in the adult
has improved rapidly, owing to gene-targeting studies that modify regulatory
pathways that affect HSCs (Adams et al.,
2006; Arai et al.,
2004; Calvi et al.,
2003; Wilson et al.,
2004; Zhang et al.,
2003). However, much work remains to be carried out in order to
define the niches and signals that dictate specific stages of HSC development
during fetal life.

This review follows the journey of developing HSCs through the fetal
hematopoietic sites and focuses on the identification of the cells, their
niches and the micro-environmental signals that are required to establish the
pool of self-renewing HSCs for life.

Establishment of definitive hematopoietic stem cell (HSC) pools in mouse
and human embryos. (A) Hematopoietic development starts as
specification of primitive streak mesoderm (gray) into hematopoietic and
vascular fates. Nascent HSCs undergo a maturation process (blue) that allows
them to engraft, survive and self-renew in future hematopoietic niches.
Subsequently, fetal HSCs expand rapidly, after which a steady state is
established in which HSCs reside in a relatively quiescent state in the bone
marrow. (B) The ages at which mouse and human hematopoietic sites are
active. Gray bars, mesoderm; red bars, active hematopoietic differentiation;
yellow bars, HSC genesis; blue bars, presence of functional adult-type HSCs.
Broken yellow bars for yolk sac and placenta indicate that de novo HSC genesis
has not been experimentally proven.

The roadmap from mesoderm to hematopoietic fate

A long-standing hypothesis posits that hematopoietic cells originate from
the hemangioblast, a mesodermal precursor cell that gives rise to blood and
endothelial cells (Sabin,
1917). The existence of a clonal precursor that has both
hematopoietic and vascular potential was first demonstrated in vitro by
differentiating mouse ES cells (Choi et
al., 1998; Kennedy et al.,
1997). These colors identified a precursor that develops between
days 2.5-4 of embryoid body (EB) differentiation and forms blast-like colonies
(named blast colony forming cell, BL-CFC). These precursors give rise to
primitive and definitive hematopoietic, endothelial and vascular smooth muscle
cells. All BL-CFCs were found among the brachyury+ Flk1+
(Kdr+; kinase insert domain protein receptor) cells, indicating
that they represent a specialized subset of mesoderm
(Fehling et al., 2003)
(Table 1), whereas subsequent
commitment into hematopoietic fate is driven, and marked by, expression of
Scl/Tal1 (T-cell acute lymphocytic leukemia 1)
(Chung et al., 2002;
D'Souza et al., 2005;
Porcher et al., 1996;
Shivdasani et al., 1995).
Importantly, Flk1+ brachyury+ hemangioblasts were also
found in the gastrulating mouse embryo
(Huber et al., 2004); however,
surprisingly few of the BL-CFCs that displayed both hematopoietic and vascular
potential were found in the yolk sac. Instead, most of them were in the
posterior primitive streak. Yet the ability to form primitive erythroid cells
suggested that these hemangioblasts were precursors of yolk sac hematopoietic
cells. These results imply that the first stages of hematopoietic development
take place before the cells migrated into the yolk sac, prior to the formation
of the blood islands, which consist of developing primitive red cells and
endothelial cells adjacent to visceral endoderm
(Palis and Yoder, 2001). This
model conflicts with a longstanding hypothesis that hemangioblasts reside in
the yolk sac and form both hematopoietic and endothelial cells within a blood
island. The migration of hematopoietic precursors from the primitive streak to
yolk sac was shown to depend on vascular endothelial growth factor signaling
through Flk1, as Flk1-knockout cells in chimeric mouse embryos
accumulate in the amnion (Shalaby et al.,
1997; Shalaby et al.,
1995). Further studies have shown that specification of
hematopoietic fate in the yolk sac depends on visceral endoderm and on signals
from Ihh (Indian hedgehog) (Baron,
2001; Dyer et al.,
2001) and Bmp4 (bone morphogenetic protein 4)
(Sadlon et al., 2004),
demonstrating the importance of a proper micro-environment for hematopoietic
commitment.

It remains to be shown whether all yolk sac hematopoietic cells and the
definitive HSCs that emerge in the embryo, and possibly the placenta,
originate from hemangioblasts. Indeed, multiple pathways might specify
hematopoietic cells from mesoderm. The inherent differences between
hematopoietic programs in the different sites, i.e. the ability of the yolk
sac to generate primitive erythroid cells, and the delay in lymphoid
potential, suggest that microenvironmental cues in sites of early
hematopoietic specification may later dictate the ultimate output of the
different hematopoietic programs. The developmental stage at which the
transient embryonic and definitive adult hematopoietic programs first diverge
at the molecular level has yet to be defined.

Assays for fetal hematopoietic stem cell (HSC) development.
(A) (Top panel) An E10.5-11 mouse embryo. HSCs can be reliably assayed
only by transplanting them into myeloablated (e.g. irradiated) recipients
(lower panel) and then documenting their ability to give rise to sustained
(3-6 months) multilineage hematopoietic reconstitution. Functional adult-type
HSCs that can achieve such reconstitution in this assay are found only in
mouse embryos or extra-embryonic tissues after E10.5-11.5. (B) (Top
panel) A mouse embryo at E9.0. HSC activity can be observed at a younger age,
and higher hematopoietic chimerism from immature HSCs (pre-HSCs) can be
obtained if more permissive recipients (e.g. newborn mice,
Rag2-/- γc-/- mice, lower left)
are used. Transplantation directly into the long bones of a recipient can also
improve engraftment (lower right). Intermediate explant culture of pSP cells
also allows detection of HSC activity at a younger age.

The yolk sac as a source of embryonic blood cells

In mice, the yolk sac is essential for the development of the embryo as it
provides the initial feto-maternal transport system before the placenta is
formed, and is the source of the first blood cells
(Ferkowicz and Yoder, 2005;
Li et al., 2003;
McGrath and Palis, 2005). It
produces the first primitive erythroid cells that enter the circulation and
the later definitive progenitors destined for the liver (reviewed by
McGrath and Palis, 2005). Both
hematopoietic progenitor populations originate within a well-defined region in
the proximal yolk sac (Ferkowicz et al.,
2003; McGrath et al.,
2003). Primitive erythrocytes develop from precursors that are
present in the yolk sac between E7.0 and 8.25, and express low levels of Cd41
(GpIIb, integrin α 2b; Itga2b - Mouse Genome Informatics). The hallmarks
of the primitive erythroid lineage are retention of the nucleus while entering
circulation, a large size, and the expression of both embryonic and adult
globins (Ferkowicz et al.,
2003; Palis et al.,
1999). However, recent studies have shown that primitive
erythrocytes enucleate in the embryonic circulation, concomitantly
downregulating βH1-globin transcripts, while activatingϵγ
-globins and upregulating adult β-chains
(Kingsley et al., 2006;
Kingsley et al., 2004).

The definitive myeloerythroid progenitors develop in the mouse yolk sac
slightly after primitive erythroid precursors
(Cumano et al., 1996;
Palis et al., 1999). These
progenitors can be first identified by expression of Cd41 and Kit (receptor
for stem cell factor), whereas the pan-hematopoietic marker Cd45 (Ptprc;
protein tyrosine phosphatase receptor C) is turned on during their progressive
maturation (Mikkola et al.,
2003). As the yolk sac microenvironment does not support terminal
differentiation into definitive blood cell lineages, these progenitors seed
the fetal liver where they generate blood cells for the growing embryo. The
definitive red cells that are derived from these precursors are smaller than
primitive erythroid cells, express only adult globins and enucleate before
entering the circulation.

Despite the abundant production of hematopoietic progenitors in the yolk
sac, it is still unclear whether the yolk sac also generates HSCs. The main
data arguing against this come from explant culture studies, which show that
early yolk sac explants do not have the potential to generate adult
reconstituting HSCs or lymphoid progeny
(Cumano et al., 1996;
Cumano et al., 2001;
Medvinsky and Dzierzak, 1996)
(Fig. 3). An alternative theory
is that the yolk sac micro-environment becomes supportive for HSC development
later on, as, by E12.5, yolk sac explant cultures can support HSC expansion in
vitro (Kumaravelu et al.,
2002). Other studies have shown that intrahepatic transplantation
of yolk sac hematopoietic cells into conditioned newborn recipients or direct
transplantation into the pre-circulation yolk sacs, permits engraftment of
E9.5 or even E8.5 yolk sac cells, respectively
(Weissman et al., 1978;
Yoder et al., 1997a;
Yoder et al., 1997b).
Although these findings suggest that immaturity of the yolk sac hematopoietic
cells accounts for their inability to survive in an adult environment, their
relative contribution to adult hematopoiesis during normal development remains
unclear. It is also possible that some of the yolk sac HSCs are derived from
the vitelline artery or upper dorsal aorta, which are upstream of the yolk sac
in circulation (de Bruijn et al.,
2000). Nevertheless, as the yolk sac does not harbor a large
quantity of HSCs, it is unlikely to support large-scale expansion of HSCs in
vivo (Gekas et al., 2005).

Emergence of HSCs in the embryo proper

The first evidence that HSCs may develop in the embryo proper came from
chick-quail chimera studies, in which HSCs derived from the embryo outcompeted
yolk sac hematopoietic cells in their contribution to adult hematopoiesis
(Lassila et al., 1978;
Martin et al., 1979).
Subsequently, intra-embryonic HSCs have been documented in multiple species
(Jaffredo et al., 2005). In
all species described, the intra-embryonic HSCs develop in close association
with the ventral wall of the dorsal aorta, or in adjacent vitelline and
umbilical arteries. This region, which is first called the para-aortic
splanchnopleurae (pSP), and later becomes the aorta-gonadmesonephros region
(AGM), develops from the lateral plate mesoderm (LPM).

Hematopoietic progenitor cells appear in the pSP-region of mouse embryos by
E8.5, yet functional HSCs that can reconstitute adult recipients are not found
until E10.5-11.0 (Cumano et al.,
1996; Godin and Cumano,
2002; Jaffredo et al.,
2005; Medvinsky and Dzierzak,
1996; Muller et al.,
1994) (Figs 2,
3;
Table 1). Notably, at E10.5,
the repopulation activity of AGM-derived HSCs in standard adult reconstitution
assays is very low, whereas their transplantation into recombination
activating gene 2 (Rag2)-/- common γ chain
(γc)-/- mouse recipients, which are deficient in B,
T and natural killer cells, allows hematopoietic reconstitution at an earlier
age and at a higher level (Cumano et al.,
2001) (Fig. 3,
Table 1). It was, thus,
hypothesized that lack of major histocompatibility complex class 1 molecule
expression in nascent HSCs may make them targets for natural killer cells.
Furthermore, transplantation of E10.5 HSCs directly into the long bones
improves hematopoietic reconstitution, suggesting that the homing and
engraftment properties of young HSCs are poorly developed at this stage
(Matsubara et al., 2005). When
AGM cells are transplanted into conditioned newborn recipient mice, in which
the liver is still an active hematopoietic organ, HSC activity could be
detected at E9.5 (Kumano et al.,
2003) (Fig. 3,
Table 1), leading to the
speculation that younger microenvironments are more supportive of fetal HSCs.
Importantly, transplantable HSCs could be generated from the pSP region of
precirculation embryos (E8.5) when the tissues were cultured as whole tissue
explants, indicating that intra-embryonic HSCs develop in situ, rather than
being imported via the circulation (Cumano
et al., 2001) (Fig.
3, Table 1).
Furthermore, stromal cells lines derived from the AGM and surrounding regions
support HSCs in culture (Kusadasi et al.,
2002; Oostendorp et al.,
2002; Oostendorp et al.,
2005). These data suggest that the pSP-AGM region is a source of
HSCs, but then a maturation process is required to confer on them the
engraftment and self-renewal abilities in the adult bone marrow
micro-environment. As the phenotype of developing HSCs changes during this
process, it is crucial to define the means to identify, localize and purify
developing HSCs at all stages in order to ultimately define the molecular
mechanisms of HSC maturation.

As developing HSCs share specific characters with endothelial cells, such
as surface marker expression and the ability to incorporate acetylated LDL
(low density lipoprotein) (Sugiyama et
al., 2003), it has been speculated that HSCs emerge from a subtype
of endothelial cells, called hemogenic endothelium
(Jaffredo et al., 2005).
Furthermore, histological sections have revealed hematopoietic clusters
budding from the ventral wall of the dorsal aorta and adjacent large vessels.
Alternative hypotheses suggest that the intra-embryonic HSCs originate from
the mesoderm/mesenchyme adjacent to the aorta
(North et al., 2002) or from
subaortic patches (Bertrand et al.,
2005), and migrate through the endothelium into the lumen of
vessels, concomitantly maturing to definitive HSCs. Indeed, individual cells
in sub-aortic patches express surface markers that are characteristic of early
hematopoietic precursors/immature HSCs [e.g. Cd41+,
Kit+, Cd45-, Cd31+, Aa4.1+
(Cd93)], and display the potential to reconstitute sublethally irradiated
Rag2-/- γc-/- mouse recipients.
These results imply that hematopoietic specification in the pSP/AGM region may
occur before precursors cells contact the aortic endothelium.

Mice in which the Runx1 (runt-related transcription factor 1) has
been targeted (which encodes a transcription factor that is essential for
definitive hematopoiesis) have been instrumental in defining and visualizing
the genesis of HSCs (North et al.,
1999; North et al.,
2002; North et al.,
2004; Okuda et al.,
1996). Runx1 is expressed in HSCs from the emergence of HSC
precursors throughout ontogeny, affording the opportunity to identify
candidate HSCs by, for example, studying Runx1-lacZ mouse embryos, in
which lacZ has been targeted into the Runx1 locus
(Fig. 4). Interestingly,
Runx1-lacZ cells localize both to the endothelium and mesenchyme
adjacent to the large vessels. Although not all Runx1-expressing
cells are likely to be HSCs, it has been suggested that the
Runx1-expressing cells in aortic mesenchyme harbor precursors for
HSCs that subsequently passage through the endothelium and are released into
the circulation (Fig. 4).
However, Runx1 haploinsufficiency leads to changes in HSC kinetics
and surface markers, such as abnormal HSC distribution and a delay in
upregulating Cd45 expression (Cai et al.,
2000; North et al.,
2002). Thus, it remains to be shown whether the mesenchymal
distribution of Runx1-lacZ cells reflects normal HSC development.
Interestingly, Runx1-lacZ knockout embryos, which die by E12.5
because of lack of definitive hematopoietic cells, also express lacZ
in cells in the mesenchyme adjacent to the dorsal aorta from E10.5 onwards.
This expression could mark the precursors that attempt to generate HSCs but
are unable to do so in the absence of functional Runx1 protein
(North et al., 1999).

Role of the placenta in establishing a definitive HSC pool

The placenta is essential for fetal development as it is responsible for
feto-maternal exchange from midgestation and produces important hormones and
cytokines (Cross et al., 2003;
Rossant and Cross, 2001).
Although the placenta has not been considered to be a hematopoietic organ,
early reports suggested that the mouse placenta may exhibit hematopoietic
activity (Dancis et al., 1977;
Dancis et al., 1968;
Melchers, 1979;
Till and McCulloch, 1961).
Studies in avian embryos have also revealed that the allantois, a
mesoendodermal appendage that functions as an excretory and respiratory outlet
for the embryo, generates hematopoietic cells that, in quailchick grafting
studies, seed the bone marrow (Caprioli et
al., 1998; Caprioli et al.,
2001). In mice, the allantois forms the umbilical vessels and the
mesodermal parts of the fetal placenta
(Downs and Harmann, 1997). It
develops after gastrulation as an extension of the posterior primitive streak
and fuses with the chorionic plate by E8.5, whereafter the allantoic mesoderm
interdigitates into the trophoblast layer of the placenta giving rise to the
fetal vasculature in the placental labyrinth. Within the labyrinth, fetal and
maternal blood spaces come into close proximity, facilitating the exchange of
gases and nutrients. Although the umbilical artery, which connects the dorsal
aorta to the placenta, has been suspected to generate HSCs, HSC activity in
the placenta has not been comprehensively studied until recently.

Hematopoiesis in the mouse placenta is evident from E9.0, when definitive
multi-lineage progenitors appear
(Alvarez-Silva et al., 2003),
whereas mature HSCs are found 1.5-2.0 days later
(Gekas et al., 2005;
Ottersbach and Dzierzak,
2005). Placental HSCs are derived from fetal cells, and fulfil the
most stringent functional criteria for adult type HSCs
(Fig. 3). The early onset of
HSC activity in the placenta, which is evident before HSCs are found in the
fetal circulation or liver, suggests that placental HSCs may be generated in
situ. If the placenta produces HSCs, a possible origin is the allantoic
mesoderm. Although hematopoiesis has not yet been documented to occur in the
early mouse allantois, this possibility needs to be investigated by assays
that detect nascent hematopoietic precursors at an earlier stage of
development than can conventional in vitro or in vivo hematopoietic assays
(Fig. 3,
Table 1). If allantoic mesoderm
proves to be the source of placental HSCs, the placenta might generate HSCs by
mechanisms similar to those in the pSP-AGM region, thereby extending the
hematopoietic activity in the dorsal aorta and the large vessels to a larger
anatomical area. Indeed, grafting experiments with genetically marked
allantoises have shown that the allantoic mesoderm and the LPM that forms the
AGM region have a similar developmental potential, in contrast to the paraxial
mesoderm that gives rise to the somites
(Downs and Harmann, 1997).
However, vascularization of the allantois proceeds from the distal tip to the
base, rather than by angiogenic sprouting from the dorsal aorta
(Downs et al., 2004),
supporting an independent origin of vascular cells, and possibly also of
hematopoietic cells, from the allantoic mesoderm.

A striking feature of placental HSCs is the rapid expansion of the HSC pool
between E11.5 and 12.5. As a result, the placenta harbors over 15-fold more
HSCs than does the AGM region or the yolk sac, suggesting that the placenta
may provide a unique microenvironment for HSC development
(Gekas et al., 2005). The
growth of the placental HSC pool may have various explanations, such as
sustained production of HSCs in the placenta, symmetric selfrenewal of
placental HSCs or maturation of nascent HSC precursors into transplantable
HSCs. Importantly, the expansion of the HSC pool during E11.5-12.5 was more
pronounced than the relative expansion of the clonogenic progenitor pool,
suggesting that the placenta micro-environment supports HSCs without
concomitant differentiation into downstream progeny
(Gekas et al., 2005).

It is also possible that HSCs originating from the AGM contribute to the
expansion of the placental HSC pool. As the main vascular route by which the
blood cells from the dorsal aorta circulate into the fetal liver goes through
the umbilical vessels and the placenta
(Sadler, 2006), this is also
the most likely route for AGM-derived HSCs take
(Fig. 5). Thus, it is possible
that AGM HSCs are nurtured temporarily in the placental niches prior to
seeding the fetal liver. Interestingly, placental HSC activity is increasing,
HSCs accumulate in the fetal liver. After E13.5, however, the number of HSCs
in the placenta decreases, while the liver HSC pool continues to expand
(Gekas et al., 2005). As the
fetal liver is directly downstream of the placenta in fetal circulation, the
placenta may supply a major fraction of the HSCs that seed the liver
(Fig. 5).

Placental HSCs have a similar surface phenotype to fetal liver HSCs, both
of which express Cd34, Kit and Sca1 at E12.5
(Gekas et al., 2005;
Ma et al., 2002;
Ottersbach and Dzierzak,
2005). However, despite the phenotypic similarity of placental and
fetal liver HSCs, the midgestation placenta is not occupied by numerous
single-lineage progenitors and definitive erythroid intermediates (as is the
fetal liver, which actively promotes erythropoiesis)
(Gekas et al., 2005). However,
it remains possible that the placenta functions as a fetal lymphoid organ, as
it has been shown that B-cell precursors appear in the mouse placenta before
they are found in the fetal liver
(Melchers, 1979). Cells that
have the potential to generate Blymphoid cells in plaque-forming assays were
found in the placenta as early as E9.5; their number peaked at E12.5 and then
declined, displaying very similar kinetics to those observed for placental
HSCs (Gekas et al., 2005). It
remains to be shown whether this reflects the presence of HSCs in the
placenta, or whether differentiation into Blymphoid cells occurs during their
residence in the placenta.

Migratory and circulatory routes that connect fetal hematopoietic
sites. The primitive streak (gray) gives rise to the hemogenic
mesoderm/hemangioblasts that migrate into the yolk sac (yellow), paraaortic
splanchnopleurae (pSP)/aorta-gonadmesonephros (AGM) region (green) and
possibly through the allantois to the placenta (blue). Hematopoietic
specification most probably occurs during the migratory process. The two main
circulatory routes that connect fetal hematopoietic organs during midgestation
are vitelline and umbilical circuits. The vitelline artery connects the upper
dorsal aorta to the yolk sac, which connects to the fetal liver (red) via the
vitelline vein. The umbilical artery connects the caudal part of dorsal aorta
to the placenta, which connects to the liver via the umbilical vein. Although
budding into the lumen and seeding through circulation has been hypothesized
as the main route by which nascent HSCs seed the fetal liver, direct migration
of hematopoietic stem cells (HSCs) from the AGM to the fetal liver has not
been formally excluded (indicated by `?'). Broken arrows indicate the
migration of HSC precursors and unbroken arrows indicate circulation of HSCs
through vasculature. Bone marrow (orange) is seeded by HSCs before birth.
Larger black arrows indicate major HSC trafficking. The timing of these events
is outlined by the timescale in embryonic days below.

Little is known about the cellular niches that support HSCs in the
placenta, or about the signals involved in their maturation, expansion and
mobilization. When the placental HSC pool grows, the placenta is hypoxic,
consistent with the notion that HSCs develop in hypoxic environments, and the
signals that drive vasculogenesis are shared with developing HSCs.
Furthermore, many signaling molecules and cytokines that have been linked to
HSC biology are expressed in the extra-embryonic ectoderm/placental
trophoblasts or allantoic mesoderm/endothelial cells
(Calvi et al., 2003;
Clemens et al., 2001;
Fujiwara et al., 2001;
Hattori et al., 2002;
Sibley et al., 2004;
Zhang and Lodish, 2004).
Further functional studies are required to identify the crucial niche cells
and molecular cues that support HSC development in the placenta.

Fetal liver in supporting HSC expansion and differentiation

The fetal liver is the primary fetal hematopoietic organ and the main site
of HSC expansion and differentiation. However, it does not produce HSCs de
novo, but is believed to be seeded by circulating hematopoietic cells
(Houssaint, 1981;
Johnson and Moore, 1975). The
first phase of fetal liver seeding initiates at E9.5-10.5, as the liver
rudiment becomes colonized by myeloerythroid progenitors that generate
definitive erythroid cells. This first wave of hematopoietic seeding most
probably derives from the yolk sac, which has prepared numerous definitive
progenitor cells and establishes the first vascular connections to the fetal
liver through vitelline vessels (Fig.
5).

The first HSCs appear in the fetal liver at E11.5. Although not
experimentally proven, it is likely that majority of the HSCs colonizing the
liver derive from the AGM and the placenta via the umbilical vessels, which is
the second major vascular circuit that connects to the fetal liver
(Fig. 5). After E12.5, the
fetal liver becomes the main fetal organ where HSCs undergo expansion and
differentiation. The number of HSCs reaches a maximum of ∼1000 HSCs by
E15.5-16.5, after which it reaches a plateau and starts to decline
(Ema and Nakauchi, 2000;
Gekas et al., 2005;
Morrison et al., 1995). At all
times, the fetal liver is rich in single-lineage progenitor cells, consistent
with its important role in producing differentiated blood cells. As gestation
proceeds, the main focus of hematopoietic differentiation changes. The early
fetal liver is rich in CFU-Es (colony-forming unit erythroid) and
proerythroblasts, reflecting active definitive erythropoiesis, whereas myeloid
and lymphoid progenitors accumulate with developmental age. It is conceivable
that the fetal liver microenvironment is modified during fetal development in
order to meet the changing needs of lineage differentiation and HSC expansion.
Conversely, the difference in fetal liver hematopoietic profile during mid-and
late gestation may reflect the lifespan of the precursor cells that originally
seed the organ, as mouse models that target transient fetal hematopoietic
populations have shown that the early fetal liver is occupied by a progenitor
population that may not contribute to adult hematopoiesis
(Emambokus and Frampton, 2003;
Li et al., 2005).

Little is known about the fetal liver niches that support HSC expansion and
differentiation. Interestingly, the HSC pool expands rapidly in the fetal
liver, whereas in the bone marrow, most HSCs are quiescent. Indeed, fetal
liver HSCs are actively cycling, and outcompete adult bone marrow HSCs when
transplanted into irradiated recipients
(Harrison et al., 1997;
Morrison et al., 1995;
Rebel et al., 1996a),
suggesting that inherent differences exist between fetal and adult HSCs.
Conversely, the fetal liver microenvironment may also provide signals that
promote symmetric self-renewing divisions of the fetal HSC pool. Investigators
have attempted to define the key components of the fetal liver
micro-environment that support HSC expansion by establishing stroma cell lines
from midgestation mouse liver cells
(Hackney et al., 2002). By
comparing the expression profiles of fetal liver-derived stroma cell lines
with different abilities to support expansion of HSCs, multiple differentially
expressed genes have been identified that probably play an important role in
the in vivo fetal liver HSC niche. The functions of the individual
differentially expressed genes in HSC biology have yet to be defined.

Another study showed that Cd3+Ter119(Ly76)- cells in
the liver can support HSC expansion in co-culture
(Zhang and Lodish, 2004).
Although the identity of the supportive cells is unknown, gene expression
analyses identified Igf2 (insulin-like growth factor 2) and angiopoietin-like
proteins as key molecules secreted by these cells
(Zhang et al., 2006;
Zhang and Lodish, 2004). When
applied alone, or in combination, Igf2 and angiopoietin-like proteins support
HSC expansion and/or survival in culture. It remains to be shown whether the
fetal liver Cd3+ cell population and the secreted factors are unique to fetal
liver microenvironment, or are used in HSC niches throughout ontogeny.

Establishing homeostasis in bone marrow HSC niches

The skeletal system develops during the third week of mouse gestation,
concomitantly establishing a unique micro-environment for HSCs within the bone
marrow. Skeletal development begins at E12.5 as mesenchymal condensations, in
which mesenchymal cells first give rise to chondrocytes that create a
cartilaginous framework for the skeleton
(Olsen et al., 2000).
Chondrocytes are later replaced by osteoblasts that generate calcified bone
through endochondral ossification. Vascular invasion into the developing bones
facilitates circulation through the bones, and the seeding of hematopoietic
progenitor and stem cells. Clonogenic progenitor activity in the long bones
starts at E15.5, whereas functional HSCs are found from E17.5 onwards
(Christensen et al., 2004;
Gekas et al., 2005). As HSCs
can be found in circulation several days before, delay in HSC colonization
implies that the early fetal bone marrow micro-environment is unable to
attract HSCs and support their engraftment and self-renewal. Sdf1 (stromal
cell-derived factor 1), a ligand for chemokine (C-X-C motif) receptor 4
(Cxcr4), is an important chemokine in bone marrow stromal cells that attracts
HSCs to fetal bone marrow, whereas it is largely dispensable for the formation
of the fetal liver HSC pool (Ara et al.,
2003). Much remains to be carried out to elucidate how the switch
from fetal liver to bone marrow hematopoiesis occurs.

Significant progress has been made in understanding the composition of HSCs
niches in the adult bone marrow (reviewed by
Wilson and Trumpp, 2006).
Mice with reduced Bmpr1a (bone morphogenetic protein receptor 1) signaling, or
constitutive expression of Pth (parathyroid hormone)/Pth-related protein
receptor in the bone, exhibit increased osteoblast and HSC numbers, leading to
the identification of osteoblasts as niche cells for HSCs
(Calvi et al., 2003;
Zhang et al., 2003).
Furthermore, recent studies suggest HSCs recognize Ca2+ upon
engraftment into the endosteal surface of bone, as Casr
(calcium-sensing receptor) knockout mice fail to establish proper bone marrow
hematopoiesis (Adams et al.,
2006). This is despite the fact that the HSC pool in the mutant
fetal liver develops normally, and the ability of HSCs to home initially to
the bone marrow remains unaffected.

Another important mechanism that regulates HSC and niche interactions is
angiopoietin signaling. Angiopoietin 1, which is secreted by the osteoblasts,
interacts with its receptor, Tie2/Tek (endothelial-specific receptor tyrosine
kinase), expressed on the surface of HSCs, and promotes the adherence,
quiescence and survival of HSCs (Arai et
al., 2004). Homotypic interactions between N-cadherin on the
surface of the osteoblasts and HSCs might also be important in anchoring HSCs
to the endosteal surface (Arai et al.,
2004). Further evidence of the importance of N-cadherin in niche
interactions has come from studies of conditionally targeted Myc
mouse knockouts (Wilson et al.,
2004). Loss of Myc in bone marrow HSCs leads to a drastic
expansion of the HSC pool, while differentiation is severely impaired,
pinpointing a central role for Myc in regulating the balance between HSC
self-renewal and differentiation. Loss of Myc is accompanied by the
upregulation of N-cadherin and integrins on the surface of HSCs, indicating
that these adhesion molecules are instrumental in anchoring the HSC to the
niche, which prevents their differentiation
(Wilson et al., 2004).

Improved HSC purification strategies have also facilitated the
identification of HSC niches in greater detail. By localizing HSCs with a
combination of SLAM-family markers
(Cd150+Cd244-Cd48-Cd41-), another
putative niche for adult HSCs has been identified in the sinusoidal
endothelium of the bone marrow and spleen
(Kiel et al., 2005;
Yilmaz et al., 2006).
Interestingly, HSCs were frequently located in the bone marrow in contact with
endothelium, whereas only a fraction localized to the endosteum. Furthermore,
in mice in which HSCs were mobilized from bone marrow by
cyclophosphamide/G-CSF (granulocyte-colony stimulating factor) treatment
(which leads to extramedullary hematopoiesis in the spleen), HSCs were mainly
found in association with sinusoidal endothelium within parafollicular areas
of the red pulp. No differences were evident in the cell cycle status between
the HSCs that were associated with the endosteum or sinusoidal endothelium in
the bone marrow. Further studies will be required to elucidate the specific
role of the different adult HSC niches.

The shift from a proliferative fetal HSC to a quiescent conservatively
self-renewing adult HSC is accompanied by distinct changes in surface marker
expression. A characteristic feature of quiescent adult HSCs is the
downregulation of Cd34, which occurs by 10 weeks of mouse development,
probably reflecting the establishment of homeostatic steady-state in bone
marrow hematopoiesis (Ogawa et al.,
2001) (Table 1).
Furthermore, changes in Cd34 expression are reversible, as Cd34-
HSCs may upregulate Cd34 expression upon activation after 5-fluorouracil
(5-FU) treatment, or mobilization into peripheral circulation by G-CSF
(Ogawa, 2002). Another
pronounced difference is downregulation of specific lineage markers from adult
HSCs. Although adult HSCs are defined by a lack of expression of lineage
commitment markers, fetal HSCs can express selected lineage markers, such as
the monocyte/macrophage marker Mac1 (Cd11b), and B-cell marker Aa4.1
(Jordan et al., 1995;
Jordan et al., 1990;
Morrison et al., 1995;
Rebel et al., 1996b)
(Table 1). It has yet to be
determined whether these changes reflect the promiscuous expression of these
markers during fetal development, or whether these molecules are
differentially required in HSCs during fetal and adult life. HSCs also change
their surface phenotype during culture, adding yet more complexity to their
identification and purification (Zhang
and Lodish, 2005).

Trafficking of HSCs during development and steady state

The development of an intact circulatory system is a prerequisite for the
survival and growth of the embryo, and for the establishment of the definitive
hematopoietic system. Before circulation is established, mesodermal precursors
migrate from the primitive streak to future hematopoietic sites, i.e. the yolk
sac, the AGM and possibly the allantois/placenta
(Fig. 5). The vascular system
develops concomitantly, and once the heart starts to beat around E8.5,
vitelline circulation is established, connecting the dorsal aorta, the yolk
sac and the heart. Although this is considered to be the onset of circulation,
mainly primitive red cells enter the bloodstream at this point. However, the
definitive hematopoietic progenitors predominantly reside in the yolk sac
until E9.25 (Palis et al.,
1999), and become distributed freely within the vascular system
only after E10.5 (McGrath et al.,
2003). By this time, the vitelline vessels have penetrated through
the liver rudiment, facilitating initial hematopoietic seeding of the fetal
liver (Fig. 5).

The second major vascular route, the umbilical circuit, develops shortly
thereafter. Umbilical vessels form from the allantoic mesoderm after
chorioallantoic fusion, and connect the dorsal aorta to the placental
labyrinthine vascular network and subsequently to the fetal liver
(Fig. 5). The development of
umbilical and labyrinthine vasculature is essential for embryonic development,
as revealed by knockout mice that exhibit placental defects and subsequent
embryonic lethality (Rossant and Cross,
2001). As the placenta is placed in a strategically favorable
position between the dorsal aorta and the fetal liver, this positioning might
ensure that HSCs, which may still be primitive in their adhesive properties,
efficiently seed the next anatomical niche that supports their development. It
is likely that the interactions between the developing HSCs and hematopoietic
niches become more specialized towards the end of gestation, as hematopoietic
niches are refined. Indeed, some mutant mouse strains, such as the
Casr knockout mouse are less able to establish bone marrow
hematopoiesis, whereas fetal liver hematopoiesis is relatively unaffected
(Adams et al., 2006). However,
the mechanisms that initially attract fetal HSCs to establish their residence
in the fetal liver and promote their exit and seeding of the bone marrow
remain unknown.

Although circulating blood harbors many hematopoietic progenitors from
midgestation, definitive HSCs appear later in circulation than in the AGM, the
placenta and the yolk sac, and are always rare compared with other circulating
hematopoietic cells (Christensen et al.,
2004; Gekas et al.,
2005; Kumaravelu et al.,
2002). Thus, although HSCs circulate during both fetal and adult
hematopoiesis, and their ability to do so is essential for HSC pool
establishment and maintenance, HSCs appear to reside most of the time in
secure hematopoietic niches. Indeed, in the adult mouse, HSCs disappear from
circulation 1-5 minutes after transplantation, demonstrating extremely rapid
homing to target hematopoietic niches
(Wright et al., 2001).
Approximately 100 HSCs are in circulation during normal homeostatic conditions
(Wright et al., 2001).
Studies using parabiotic mice, where two individual animals are connected
through circulatory systems, and using consecutive transplantations into
unconditioned immunodeficient mice, suggest that a small number of HSC niches
are freed constantly and can be filled by circulating HSCs, while the exiting
HSCs establish residence in another anatomical location
(Bhattacharya et al., 2006;
Cao et al., 2004;
Wright et al., 2001).

Concluding remarks

The importance of stem cell micro-environments has been only recently fully
appreciated. Yet, much can be learned from the biology of HSCs by studying the
niches that support them. Although many common signals may be used in fetal
and adult HSC niches, distinct regulatory mechanisms exist that promote the de
novo generation or expansion of HSCs during fetal life, or support the
relative quiescence of HSCs during steady state hematopoiesis in the adult.
This is exemplified by differential requirements for transcription factors
during specific stages of HSC development
(Teitell and Mikkola, 2006),
and by inherent differences between fetal and adult HSCs, as shown by their
predisposition to undergo expansion or quiescence, and by their different
surface marker profiles. These inherent differences should not be overlooked,
but may instead be used to identify a subtype of HSCs that is more likely to
retain self-renewal ability during therapeutic manipulations. The challenge is
to dissect the hematopoietic niches in various anatomical locations into
specific cellular and molecular components that direct each stage of HSC
development, and translate that knowledge into the tools that can be used to
culture or manipulate HSCs. A key factor that limits the availability of HSCs
for bone marrow transplantation is the difficulty to sustain their
self-renewal capacity and multipotency ex vivo. Understanding how these
programs are established and maintained during development will be
instrumental for developing better culture systems. Ultimately it should be
possible to learn how to recapitulate the correct micro-environment for ex
vivo expansion of HSCs derived from cord blood, adult bone marrow or mobilized
peripheral blood, or even for the generation of long-term reconstituting HSCs
from novel HSC sources, such as human ES cells
(Lerou and Daley, 2005). These
advances will ultimately improve HSC-based therapies for blood cell disorders
such as hematopoietic malignancies, and inherited anemias and
immunodeficiencies (Bordignon,
2006).

Acknowledgments

H.K.A.M.'s research was supported by the NIH and Harvard Stem Cell
Institute Seed Grant.

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